Analogs of Coenzyme A (CoA) and CoA esters modified in the thiol/thioester moiety are valuable for studies of enzymes which use CoA or CoA esters as substrates. Methods are being developed for using the enzymes catalyzing the last two steps in Coenzyme A (CoA) biosynthesis as catalysts in the synthesis of CoA analogs. These reactions employ chemically synthesized pantetheine phosphate analogs as alternate substrates. This strategy is being used for the synthesis of CoA analogs of potential value as enzyme inhibitors, mechanistic probes, and as affinity ligands for chromatography. In addition, truncated CoA aldehydes will be prepared for activating simple thioesters as substrates for CoA ester utilizing enzymes via a reversible linkage strategy. This strategy will be useful in a wide range of acyl transfer and asymmetric enoyl-thioester hydration reactions. This project will require mass spectrometry for the analysis and characterization of pantetheine phosphate and coenzyme A analogs. These compounds are difficult to characterize by elemental analysis due to the variable protonation states of the multiple ionic functional groups. NMR, while valuable, does not provide conclusive analysis due to the complexity of these molecules and difficulty in assignment of all resonances and does not prove elemental composition.